Project Summary/Abstract The goal of this project at True North Therapeutics is to develop monoclonal antibodies that specifically inhibit the alternative pathway of complement (AP). Inhibition of the AP at the level of the upstream amplification loop is designed to be an improvement over the current standard of care for diseases such as paroxysmal nocturnal hemoglobinuria (PNH). The current anti-C5 therapeutic eculizumab does abolish intravascular hemolysis in almost all PNH patients, eventually resulting in a substantial clinical benefit in terms of symptoms and acute disease manifestations. Nevertheless, the hematological benefit is extremely heterogeneous, since about one third (20-40%) of patients still remain transfusion-dependent or severely anemic. By inhibiting the accumulation of the upstream C3 split products that serve as opsonins, our goal is to prevent the destruction of RBCs via phagocytosis by cells of the mononuclear phagocyte system (extravascular hemolysis). In addition, we have been pursuing a strategy designed to circumvent the dosing challenges that have severely limited the field of complement therapeutics by developing an AP-specific antibody that binds only the active product of the AP pathway protein Factor B, Factor Bb (fBb). In this Phase I SBIR proposal, we seek to continue the development of two monoclonal antibodies to fBb, both of which inhibit AP-mediated hemolysis in human and cynomolgus serum. M10 is our lead antibody and shows complete specificity for the active fBb form, with no binding to full length Factor B (FB) in solution. M12 binds both fBb and FB with similar affinities, and will be used to test the hypothesis that targeting only the active form of this protein will lead to improved PK/PD. In Specific Aim 1 we will produce human chimeric forms of these antibodies (hM10 and hM12) and confirm their activity and specificity in a series of in vitro assays. In Specific Aim 2, we will continue in vitro testing using experimental conditions relevant to PNH, including both in PNH-like RBCs and in RBCs from PNH patients provided by external collaborators. In Specific Aim 3 we will test hM10 and hM12 side by side in cynomolgus monkeys to compare their performance in PK/PD studies.